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Recent advancements in DNA-based approaches have led to the identification of uncommon and rare bacterial pathogens. In this study, by utilizing a DNA-based approach, a total of 1043 clinical specimens were processed for the identification of actinobacteria targeting the 16S rRNA and gyrB genes. Drug susceptibility testing was also conducted using micro-broth dilution and PCR. Two isolates of Nocardia flavorosea and Rhodococcus erythropolis were reported for the first time in Iran. Also, Nocardiopsis dassonvillei, Streptomyces olivaceus, and Streptomyces griseus were reported for the first time in Asia. Infections caused by Nocardia caishijiensis and Prauserella muralis have also been reported in this study. The first Asian case of pulmonary infection caused by Nocardia ignorata and the first global case of brain abscess caused by Nocardia ninae and Nocardia neocaledoniensis have been reported in this study. Overall 30 isolates belonging to 6 genera (Nocardia, Streptomyces, Rodoccoccus, Nocardiopsis, Rothia, and Prauserella) were detected in 30 patients. All 30 isolates were susceptible to amikacin and linezolid. Three isolates including Nocardia otitidiscaviarum (n = 2) and Nocardia flavorosea (n = 1) were resistant to trimethoprim-sulfamethoxazole which were the first trimethoprim-sulfamethoxazole resistant clinical actinomycetes in Iran. Isolation of rare species of actinomycetes particularly Nocardia spp. requires urgent action before they spread clinically particularly among immunocompromised patients.

A novel actinomycete, designated strain 3C-HV12T, was isolated from the cuticle of an ant (Lasius fuliginosus L) and characterised using a polyphasic approach. 16S rRNA gene sequence similarity studies showed that strain 3C-HV12T belongs to the genus Nocardia with high sequence similarities to Nocardia soli DSM 44488T (99.2 %) and Nocardia cummidelens R89T (99.2 %), and phylogenetically clustered with these two species and Nocardia ignorata DSM 44496T (98.8 %), Nocardia salmonicida JCM 4826T (98.8 %), Nocardia fluminea S1T (98.8 %), Nocardia coubleae OFN N12T (98 %) and Nocardia camponoti 1H-HV4T (97.4 %). The morphological and chemotaxonomic properties of the strain are also consistent with those of members of the genus Nocardia. The strain was observed to form extensively branched substrate hyphae which fragmented into rod-shaped and non-motile elements. The cell wall was found to contain meso-diaminopimelic acid and the whole cell sugars were identified as arabinose and galactose. The predominant menaquinone was identified as MK-8(H4, ω-cycl). The phospholipid profile was found to consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. The major fatty acids were identified as C18:0 10-methyl, C16:0 and C16:1ω7c. Mycolic acids were found to be present. A combination of DNA–DNA hybridisation experiments and phenotypic tests were carried out between strain 3C-HV12T and its phylogenetically closely related strains, which further clarified their relatedness and demonstrated that 3C-HV12T could be distinguished from these strains. Therefore, the strain is concluded to represent a novel species of the genus Nocardia, for which the name Nocardia lasii sp. nov. is proposed. The type strain is 3C-HV12T (=DSM 100525T = CGMCC 4.7279T).

Nocardia ignorata, which was first described in 2001, is a rare human pathogen. We report a case of pulmonary nocardiosis caused by this bacterium in a 55-year-old man from Iran. The patient, a gardener, had frequent exposure to soil and may have acquired the infection from that source.

Type strains of 72 validated Nocardia species were phylogenetically analyzed based on the multilocus sequence analysis (MLSA) concatenated atpD-groL1-groL2-recA-rpoA-secY-sodA-ychF. Furthermore, their similarity based on digital DNA-DNA hybridization (dDDH) was calculated. Nocardia soli, Nocardia cummidelens and Nocardia salmonicida, Nocardia nova and Nocardia elegans, Nocardia exalbida and Nocardia gamkensis, and Nocardia coubleae and Nocardia ignorata formed coherent clades, respectively. Moreover, each set showed over 70% relatedness by dDDH and shared common phenotypic characteristics. Therefore, we propose a reclassification of Nocardia soli and Nocardia cummidelens as a later heterotypic synonym of Nocardia salmonicida, Nocardia elegans as a later heterotypic synonym of Nocardia nova, Nocardia gamkensis as a later heterotypic synonym of Nocardia exalbida, and Nocardia coubleae as a later heterotypic synonym of Nocardia ignorata.

International audience; Actinobacteria are well known for their potential in biotechnology and their production of metabolites of interest. Lichens are a promising source of new bacterial strains, especially Actinobacteria, which afford a broad chemical diversity. In this context, the culture medium of the actinobacterium Nocardia ignorata, isolated from the terrestrial lichen Collema auriforme, was studied. The strain was cultivated in a BioFlo 115 bioreactor, and the culture medium was extracted using an XAD7HP resin. Five known diketopiperazines: cyclo (l-Pro-l-OMet) (1), cyclo (l-Pro-l-Tyr) (2), cyclo (d-Pro-l-Tyr) (3), cyclo (l-Pro-l-Val) (4), cyclo (l-Pro-l-Leu) (5), and one auxin derivative: indole-carboxaldehyde (8) were isolated, along with two new brominated diketopiperazines: cyclo (d-Pro-l-Br-Tyr) (6) and cyclo (l-Pro-l-Br-Tyr) (7). Structure elucidation was performed using HRMS and 1D and 2D NMR analysis, and the synthesis of compounds 6 and 7 was carried out in order to confirm their structure.

International audience; Lichens are symbiotic organisms consisting of fungi and algae (Chlorophyta or Cyanobacteria), and are well-known as rich sources of novel compounds [1]. However, recent studies revealed the presence of bacterial microbiota associated with these organisms [2]. These microorganisms may represent an underexplored reservoir of novel species of potential interest in the discovery of novel lead compounds. However, the biosynthesis of original microbial compounds is influenced by cultivation conditions such as nutrients, incubation periods, pH, temperature, etc. Changing these parameters according to the OSMAC approach [3] (One Strain MAny Compounds) can result in different secondary metabolite profiles. Actinobacteria are known for their ability to produce compounds of clinical and pharmaceutical importance [4]. Thus, we applied the OSMAC approach to Nocardia ignorata, which was isolated from Collema auriforme [5]. The extracts obtained from various culture media (LB, TY, MB, ISP2) showed interesting cytotoxicity against two different cell lines. A statistical optimization of the production of active compound was established using Tagushi L9 orthogonal array design. The four selected parameters were temperature, pH, volume and oxygenation of the medium with three different levels for each parameter. After several days of growth using a 5L bioreactor BioFlo® 115, the culture medium TY was collected and extracted with XAD-7 resin yielding two extracts (supernatant and resin extracts). A bioguided isolation process showed that the cytotoxicity was focused in the supernatant (HaCaT (Human keratinocyte cells): IC50= 8.0 ± 1.5 µg/mL; B16 (Murine melanoma cells): IC50 = 4.6 ± 1.8 µg/mL). This extract was then purified using flash chromatography, semi-preparative reversed-phase HPLC using a water/CH3CN gradient. This process led to the isolation of an active compound which is a novel bromide diketopiperazine identified by NMR and mass spectrometry data.

Five clinical isolates of Nocardia that showed ambiguous bases within the variable region of the 16S rRNA gene sequence were evaluated for the presence of multiple copies of this gene. The type strains of three Nocardia species, Nocardia concava , Nocardia ignorata , and Nocardia yamanashiensis , which also showed ambiguous bases in the variable region, were also examined. Cloning experiments using an amplified region of the 16S rRNA that contains the variable region showed that each isolate possessed 16S rRNA genes with at least two different sequences. In addition, hybridization studies using a 16S rRNA gene-specific probe and extracted genomic DNA of the patient isolates and of the type strain of N. ignorata showed that each isolate possessed at least three copies of the gene. These multiple differing copies of the 16S rRNA gene and the results of DNA-DNA hybridization studies indicate problems of species definition and identification for such isolates. A broader species concept than that currently in vogue may be required to accommodate such organisms.

Two bacterial isolates from Kuwaiti soil contaminated by crude oil were analysed by using a polyphasic taxonomic approach. The isolates, designated OFN N11 and OFN N12T, were shown to have molecular, chemical and morphological properties typical of members of the genus Nocardia. Based on a multigenic approach that included 16S rRNA, hsp65 and sod gene sequencing, these novel isolates formed a monophyletic clade within the genus Nocardia. The closest species was Nocardia ignorata (with 99.4 %, 99.5 %, 98.6 % gene sequence similarity to the 16S rRNA, hsp65 and sod genes, respectively). The novel isolates could be distinguished phenotypically from the type strains of recognized species of the genus Nocardia. The novel isolates were not related to the type strain of N. ignorata in DNA–DNA hybridization experiments (26 % relatedness). On the basis of these genotypic and phenotypic data, the two isolates appear to represent a novel species, for which the name Nocardia coubleae sp. nov. is proposed. The type strain is OFN N12T (=DSM 44960T=CIP 108996T).

Nocardia ignorata is a recently described species identified on the basis of a single isolate of unknown origin. Here we describe the epidemiological, phenotypic, and phylogenetic characteristics of this new species, based on five new clinical and soil isolates.

Aims: The present study was conducted to identify Nocardia spp. from Iran soil by various phenotypic tests. Materials and Methods: A total of 300 soil samples were collected of five different geographical regions in Iran. Nocardia isolation was performed by paraffin baiting technique. The colonies that were similar to be Nocardia spp. were stained with Gram, partially acid fast and acid-fast. Phenotypic tests were used for identification of Nocardia spp. Results: After analysis of phenotypic tests, the identified species are as follow: Nocardia asteroides (49.12%), Nocardia cyriacigeorgica (24.56%), Nocardia otitidiscaviarum (38.6%), N. asteroides complex (19.29%), Nocardia africana (3.5%), and Nocardia pseudobrasiliensis, Nocardia coubleae, Nocardia ignorata (each species; 1.75%). In this study, some of isolates (8.77%) remained undetected. Conclusion: Due to great number of Nocardia spp., high similarities among biochemical characteristics of species and the variability of some these characters, a wide range of biochemical tests should be used to identify Nocardia spp. to gain more accurate results.